This invention relates to methods for detecting and measuring radioactivity in liquid samples and devices for detecting and measuring radioactivity in liquid samples. Particularly, the invention relates to methods of detecting and statically counting concentrations of radioisotopes, and devices for detecting and statically counting concentrations of radioisotopes.
Radioisotopes are an important tool in many industries and various types of research, including medical research. For example, radioisotopes are used to label developmental compounds to understand their pharmacokinetics and how they are metabolized in animals and humans. In agrochemical research, radioisotopes are sometimes an important tool for complete understanding of the chemical behavior in the environment. Common radioisotopes used in research include 14C and 3H.
High performance liquid chromatography (HPLC) is widely used for detection and quantifying of radioisotope compounds. Radioactivity flow-through detectors are used to detect radioactive components in an HPLC eluate containing radioisotopes and radioisotope-labeled compounds. There is constant need for improved devices and methods for detecting and quantifying radioisotopes and radioisotope-labeled compounds.
Radioactive decay of a radioisotope is a random process following a Poisson distribution where the standard deviation, xcexa3, equals the square root of the total number of times a radioactive decay was observed, or counted, during a counting experiment:
xcexa3={square root over (Total Number of Decays)}
This formula is the basis for calculating the practical lower radioactivity detection limit of a sample containing radioisotopes when combined with information about background levels of radiation. The standard deviation, A, of background radiation produced by a reference sample is expressed in the number of times a radiative decays is measured in the reference sample, i.e. disintegrations per min, xe2x80x9cDPM.xe2x80x9d The scope of radioactivity detection is then expressed using the following equation:   Ld  ∝      B          E      *      T      
wherein Ld is the lower limit of detection measured in DPM, B is the background radiation level measured in DPM, E is the counting efficiency measured as a percentage, and T is the length of time spent measuring radioactive decay, i.e. counting time, measured in minutes.
As shown by the formula, the limits of radioactivity detection can be improved by reducing the background level of radioactivity, increasing the counting efficiency of the system, and/or increasing the counting period. Improvement of any one of these parameters would lower the limit of radioactivity detection and thereby enable the measurement of lower concentrations of radioisotopes in a sample.
Several attempts have been made to increase detection sensitivity by reducing background radioactivity levels, increasing counting efficiency, and/or increasing the counting time. For example, Packard Instruments attempted to reduce the background level of radioactivity and thereby lower the limit of radioactivity detection by using time-resolved background reduction technology. Similarly, liquid scintillants can be used to replace solid scintillants for liquid cells in order to increase counting efficiency.
Conventional methods of improving detection sensitivity also involve taking fractions from a continuously running chromatography column and analyzing them in batches, i.e. taking them xe2x80x9coff-line,xe2x80x9d in order to increase counting time. Off-line methods are undesirable because they can not detect and quantitate volatile radioactive components with a very high degree of accuracy because the volatile components tend to evaporate during the fraction collection process.
Conventional methods of increasing counting time are accomplished by collecting eluate in fractions in individual vials using a fraction collector or well fraction collector. Scintillate is then added xe2x80x9coff-linexe2x80x9d so that the number of radioactive decays can be measured. This manual process is not desirable in modem laboratories because it is labor intensive, inefficient, and costly. An exemplary xe2x80x9coff-linexe2x80x9d counting process is accomplished by manually collecting fractions in a large number of well plates, such as 96 well plates. The eluate is evaporated and manually counted statically using a multi-well counter.
A stop flow apparatus for determining radioactivity of chromatographic samples is disclosed by Lee, D. Y., PCT/US98/20324 filed Sep. 25, 1998, incorporated herein by reference in its entirety.
Conventional methods have also attempted to lengthen counting time without using a fraction collector. However, these methods fail to accurately detect all radioactive components in samples that contain a number of radioactive components and where the radioactivity of each component is different. Examples of typical methods are provided by A. C. Berick (U.S. Pat. No. 4,137,451. 1981) herein incorporated by reference in its entirety, Berthold (U.S. Pat. No. 4,704,531. 1987) herein incorporated by reference in its entirety, and Dietzel (U.S. Pat. No. 5,166,513. 1992) herein incorporated by reference in its entirety.
A. C. Berick (U.S. Pat. No. 4,137,451. 1981) collects fractions in rotating devices with many tubes that contain scintillant. The tubes are then measured sequentially. Berthold (U.S. Pat. No. 4,704,531. 1987) detects radioactive peaks using a mass detector such as an ultravoilet (UV) detector and then diverts radioisotope containing fractions as they appear as peaks on the UV detector to a radioactivity detector for static counting. In addition, Diezel (U.S. Pat. No. 5,166,513. 1992) detects radioactive peaks using a monitor radioactive detector instead of using a ultraviolet detector, and then diverts radioisotope containing fractions into a secondary radioactivity detector for static counting. In Diezel""s attempt, when multiple radioisotope containing fractions elute, the second and subsequent fractions are diverted to a third radioactive detector and subsequent radioactive detectors for static counting. Diezel""s method also has the undesirable problem of increased difficulty of recognizing peaks of radioisotope containing fractions on the monitor as radioactivity levels get closer to background levels of radioactivity. Therefore, methods and devices that address these needs have long been sought.
An apparatus and method for accurate radioisotope counting in radio-LC is described. An chromatogram or a region of chromatogram is divided into multiple fractions. Each fraction is fed/delivered precisively into the transparent and effective section of the flow cell before conducting static counting. When a radioactive fraction is detected, the content of the flow cell is flushed out using either scintillant, solvent, or a gas. Thus, in some embodiments, the current invention includes following steps: a) flow, b) stop-flow, c) precise positioning of the fraction, d) static counting, and e) memory effect removal. In some embodiments Step c) can optionally be eliminated if a fine tube is positioned right at the beginning of the effective section of the flow cell to flush the flow cell with either a scintillant, solvent or gas. The sensitivity and accuracy are improved significantly.
In some embodiments of the invention, methods are provided for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a chromatography column;
(ii) a radioactivity detector having a flow cell, said flow cell having a radiation detection area;
(iii) a conduit for flowing eluate from said chromatography column into said flow cell of said radioactivity detector;
(iv) means for introducing a gas disposed in either:
1) said conduit; or
2) said flow cell; and
(v) a controlable source of said gas;
b) flowing said eluate from said chromatography column through said conduit and into said flow cell;
c) stopping said flow of said eluate;
d) counting the radioactivity of said eluate in said radiation detection area of said flow cell;
e) flushing said flow cell with said gas to remove said eluate therefrom; and
f) optionally repeating steps (b) through (e).
In some embodiments, said means for introducing said gas is disposed in said conduit. In other embodiments, said means for introducing said gas is disposed in said flow cell.
In accordance with the present invention, methods are also provided for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector having a flow cell, said flow cell having an input, an output, and a radiation detection area;
(iii) a conduit for conducting eluate from said chromatography column to said input of said flow cell, said conduit having interposed therein means for introducing an agent solution into said conduit and mixing said agent solution with said eluate, to produce a mixed eluate-agent solution; and
(v) means for introducing a gas into at least said radiation detection area to remove said eluate from said radiation detection area;
b) either:
(i) simultaneously flowing said eluate from said chromatography column and said agent solution through said introducing and mixing means, thereby flowing said mixed eluate-agent solution into said radiation detection area of said flow cell; or
(ii) flowing said eluate from said chromatography column through said introducing means and into said radiation detection area of said flow cell;
c) stopping said flow of said eluate or said mixed eluate-agent solution;
d) counting the radioactivity of said eluate in said detection area of said flow cell in a static fashion;
e) flushing at least said radiation detection area of said flow cell with said gas to remove said mixed eluate-agent solution therefrom;
f) optionally flushing said radiation detection area of said flow cell with said agent solution; and
g) optionally repeating steps (b) through (f).
Preferably, where a liquid flow cell is employed, step (b) comprises (i), and where a solid flow cell is employed, step (b) comprises (ii).
In some embodiments, said means for introducing said gas is disposed in said conduit between said mixing means and said radiation detection area. Preferably, said means for introducing said agent solution into said conduit comprises a mixing tee; and a conduit connecting said mixing tee to a source of said agent solution. In some embodiments, said means for introducing said gas is disposed in said conduit between said source of said agent solution and said mixing tee, and in other embodiments, said means for introducing said gas is disposed in said flow cell.
In some embodiments, the invention provides methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector having a solid flow cell, said flow cell having an input, an output, and a radiation detection area;
(iii) a conduit for conducting eluate from said chromatography column to said input of said flow cell, said conduit having interposed therein means for introducing an agent solution into said conduit; and
(v) means for introducing a gas into at least said radiation detection area to remove said eluate from said radiation detection area;
b) flowing said eluate from said chromatography column through said introducing means and into said radiation detection area of said flow cell;
c) stopping said flow of said eluate;
d) counting the radioactivity of said eluate in said detection area of said flow cell in a static fashion;
e) flushing at least said detection area of said flow cell with said gas to remove said mixed eluate-agent solution therefrom;
f) optionally flushing said detection area of said flow cell with said agent solution; and
g) optionally repeating steps (b) through (f).
In some embodiments, said means for introducing said gas is disposed in said conduit between said introducing means and said radiation detection area. In further embodiments, said means for introducing said agent solution into said conduit comprises:
a tee; and
a conduit connecting said tee to a source of said agent solution.
In further embodiments, said means for introducing said gas is disposed in said conduit between said source of said agent solution and said tee. In still further embodiments, said means for introducing said gas is disposed in said flow cell.
Also provided by the present invention are improvements to a stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a solution containing at least one radiolabeled species and an agent solution into a means for mixing said radiolabeled sample solution and an agent solution to produce a mixed sample-agent solution;
(b) flowing said mixed sample solution from said mixing means through a conduit and into a flow cell having a radiation detection area for detection of radioactivity;
(c) stopping said flow of said mixed sample solution;
(d) counting the radiation of the sample within said radiation detection area; and
(e) optionally flushing at least said radiation detection area with a solvent or with said agent solution;
(f) optionally repeating steps (a) through (e); the improvement comprising:
after step (d), flowing into said flow cell a volume of gas that is at least as great as the volume of said sample in said flow cell to effect removal of said sample solution therefrom.
Also provided by the present invention are improvements to stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a sample solution containing a radiolabeled species into a conduit and into a solid flow cell having a radiation detection area for detection of radioactivity;
(b) stopping said flow of said sample solution;
(c) counting the radiation of said sample within said radiation detection area; and
(d) optionally flushing at least said radiation detection area with a solvent; and
(e) optionally repeating steps (a) through (d); the improvement comprising:
after step (c), flowing into said flow cell a volume of gas that is at least as great as the volume of said sample in said flow cell.
Also provided by the invention are methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a chromatography column providing an eluate therefrom;
(ii) a radioactivity detector having a flow cell, said flow cell having a radiation detection area from which radiation is counted;
(iii) a conduit for flowing said at least said eluate from said chromatography column into said flow cell, said conduit having disposed means for introducing an agent solution into said conduit and mixing said eluate and an agent solution to form a mixture thereof;
(iv) means for introducing a gas into said flow cell; and
(v) a controlable source of said gas;
b) flowing either:
(i) said mixture of said agent solution and said eluate; or
(ii) said eluate through said conduit and into said flow cell;
c) stopping said flow of said mixture;
d) flowing a volume of said agent solution through said introducing means and into said conduit means in an amount at least equal to the total volume of said conduit, said introducing means and said flow cell preceding said radiation detecting area, to ensure that said volume of eluate or mixed agent-sample solution residing in said conduit, said introducing means and said flow cell preceding said radiation detecting area is placed in said radiation detection area;
e) counting the radioactivity in said radiation detection area of said flow cell;
f) flushing said flow cell with said gas to remove said eluate therefrom; and
g) optionally repeating steps (b) through (f);
wherein said means for introducing said gas is disposed within said radiation detection area of said flow cell. Preferably, said flow cell comprises:
i) an exterior surface and an interior surface, said interior surface defining said radiation detection area;
ii) an inlet through-aperture;
iii) an exit through-aperture; each of said inlet and exit through-apertures connecting said radiation detection area with said exterior surface;
iv) tubular means entering through said inlet through-aperture, and exiting through said exit through-aperture, and directing flow of said eluate through said radiation detection area; and
v) flushing means for flushing the majority of said eluate from said radiation detection area, said flushing means comprising a flushing tube interposed within said tubular means, said flushing means being sealingly connected at one end to a source of gas, and open at the other end; said open end residing inside said tubular means at a point within said radiation detection area of said flow cell; and
vi) means for entry of said flushing tube into said tubular means.
In some embodiments, the flow cell is a liquid flow cell, and in other embodiments, the flow cell is a solid flow cell. Preferably, where a liquid flow cell is employed, step (b) comprises (i), and where a solid flow cell is employed, step (b) comprises (ii).
In some embodiments, said tubular means is in the form of a coil. In further embodiments, the method further comprises a grounding wire interposed within said tubular means, preferably in the form of a helix.
In some embodiments, said tubular means is a thermoplastic polymer tube, preferably a teflon tube.
Some embodiments of the foregoing method further comprise the step of flushing said flow cell with a fluid, preferably after step (f).
In some embodiments of the foregoing method, said means for entry of said flushing tube is located on said tubular means at a point prior to said inlet through-aperture.
In further embodiments of the foregoing method, said flow cell further comprises a flushing line through-aperture connecting said exterior surface and said inlet through aperture, said flushing tube being disposed within said flushing line through-aperture, and wherein said means for entry of said flushing tube into said tubular means is located on said tubular means at a point within said inlet through aperture.
In still further embodiments of the foregoing method, said flow cell further comprises a flushing line through-aperture connecting said exterior surface and said interior surface, said flushing tube being disposed within said flushing line through-aperture, and wherein said means for entry of said flushing tube into said tubular means is located on said tubular means at a point within said radiation detection area.
In some embodiments, said flushing of said flow cell with said gas in step (f) comprises directing a steady stream of gas through said flushing means, or a discontinuous stream of gas through said flushing means, or pulses of gas through said flushing means.
Also provided in accordance with the present invention methods for measuring the radioactivity of an eluate from a liquid chromatography column, the method comprising:
(a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector comprising a flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iii) a conduit for conducting said eluate from said chromatography column into said flow cell of said radioactivity detector;
(iv) means for introducing an agent solution into said eluate interposed in said conduit;
b) simultaneously flowing said eluate from said chromatography column and said agent solution to produce a mixed eluate-agent solution in said conduit;
c) flowing said mixed eluate-agent solution into said flow cell;
d) stopping said flow of said eluate;
e) flowing a volume of said agent solution into said conduit that is sufficient to ensure that said mixed eluate-agent solution residing in said means for introducing said agent solution, in said conduit downstream of said means for introducing said agent solution, and in said flow cell preceding said radiation detecting region is placed into said radiation detection area of said flow cell;
f) counting the radioactivity of the solution in said radiation detection area in a static fashion;
g) optionally flushing said flow cell; and
h) optionally repeating steps (b) through (f).
Preferably, said flushing in said step (g) comprises the steps of:
i) flushing said flow cell with a first fluid;
j) optionally flushing said flow cell with a second fluid; and
k) optionally flushing said flow cell with a third fluid;
wherein each of said fluids is independently a gas, a solvent or an agent solution.
In some embodiments, said first fluid and said third fluid are each an inert gas, and said second fluid is a solvent or an agent solution, and in other embodiments said first fluid and said third fluid are each a solvent or an agent solution, and said second fluid is an inert gas. In some embodiments, the inert gas is helium or nitrogen.
In some embodiments of the foregoing methods, steps b-h are performed for every fraction eluted from said chromatography column. In other embodiments of the foregoing methods, steps b-h are performed only when a threshold amount of radioactivity is detected by said radioactivity detector.
In some embodiments of the foregoing methods, the liquid chromatograph further comprises a UV (i.e., ultraviolet)-visible absorbance detector. In some embodiments, steps b-h are performed only when a threshold amount of UV or visible electromagnetic radiation is detected by said UV-visible absorbance detector.
Also provided in accordance with the present invention are methods for measuring the radioactivity of an eluate from a liquid chromatography column, the method comprising:
(a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector comprising a flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iii) a conduit for conducting said eluate from said chromatography column into said flow cell of said radioactivity detector;
(iv) means for introducing an agent solution into said eluate interposed in said conduit;
b) flowing said eluate into said flow cell;
d) stopping said flow of said eluate;
e) flowing a volume of said agent solution into said conduit that is sufficient to ensure that said eluate residing in said means for introducing said agent solution, in said conduit downstream of said means for introducing said agent solution, and in said flow cell preceding said radiation detecting region is placed into said radiation detection area of said flow cell;
f) counting the radioactivity of the solution in said radiation detection area in a static fashion; and
g) optionally flushing said flow cell;
h) optionally repeating steps (b) through (g).
In some embodiments of the foregoing methods, said flushing in said step (g) comprises the steps of:
i) flushing said flow cell with a first fluid;
j) optionally flushing said flow cell with a second fluid; and
k) optionally flushing said flow cell with a third fluid;
wherein each of said fluids is independently a gas, a solvent or an agent solution.
In some embodiments of the foregoing methods, said first fluid and said third fluid are each an inert gas, and said second fluid is a solvent or an agent solution, and in other embodiments said first fluid and said third fluid are each a solvent or an agent solution, and said second fluid is an inert gas.
In some embodiments of the foregoing methods, steps b-h are performed for every fraction eluted from said chromatography column. In other embodiments of the foregoing methods, steps b-h are performed only when a threshold amount of radioactivity is detected by said radioactivity detector.
In some embodiments of the foregoing methods, the liquid chromatograph further comprises a UV (i.e., ultraviolet)-visible absorbance detector. In some embodiments, steps b-h are performed only when a threshold amount of UV or visible lightelectromagnetic radiation is detected by said UV-visible absorbance detector.
In some embodiments of the foregoing methods, step (b) further comprises simultaneously flowing said agent solution to produce a mixed eluate-agent solution in said conduit; and flowing said mixed eluate-agent solution into said flow cell.
Also provided by the present invention are methods for determining the radioactivity of an eluate from a liquid chromatography column, said method comprising:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) pump means for pumping solvent through said chromatography column to produce an eluate;
(iii) a radioactivity detector comprising a flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iv) a first conduit for introducing said eluate into said flow cell of said radioactivity detector;
(v) mixing means disposed in said first conduit;
(vi) a second conduit for introducing an agent solution into said mixing means;
(vi) pump means for pumping said agent solution; and
(vii) an optionally present UV-visible absorbance detector;
b) either:
(1) pumping said eluate into said radiation detector so that at least part of said radiation detection area of said flow cell is occupied by said eluate; or
(2) simultaneously pumping said eluate from said chromatography column and said agent solution through said mixing means so that at least part of said radiation detection area of said flow cell is occupied by said mixed eluate-agent solution;
c) stopping said flow of said eluate or said mixed eluate-agent solution;
d) pumping a volume of said agent solution through said second conduit and said mixing means and into said first conduit, said volume of agent solution being sufficient to ensure that said eluate or said mixed eluate-agent solution residing in said first conduit, said mixing means and said flow cell preceding said radiation detection area is placed into said radiation detection portion of said flow cell;
e) counting the radioactivity residing in the radiation detection portion of the flow cell in a static fashion;
f) flushing at least said radiation detection area of said flow cell with said agent solution; and
g) optionally repeating steps (b) through (f).
In some embodiments, the flow cell is a liquid flow cell, and in other embodiments, the flow cell is a solid flow cell. Preferably, where a liquid flow cell is employed, step (b) comprises (2), and where a solid flow cell is employed, step (b) comprises (1).
In some embodiments, said second conduit comprises means for the introduction of a gas into said second conduit. In other embodiments, said method further comprises the step of flushing at least said radiation detection area of said flow cell with said gas, said gas flushing step being performed either immediately before or immediately after step (f).
In some embodiments, said mixing means disposed in said first conduit is a mixing tee. In further embodiments, said means for the introduction of a gas into said second conduit is a tee. In further embodiments, said agent solution comprises a scintillant.
In some embodiments, steps d-g are performed for every fraction eluted from said chromatography column. In other embodiments, steps d-g are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps d-g are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided in accordance with the present invention are methods for accurately determining the radioactivity of an eluate from a liquid chromatography column, said method comprising:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) pump means for pumping solvent through said chromatography column to produce an eluate;
(iii) a radioactivity detector comprising a solid flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iv) a first conduit for introducing said eluate into said flow cell of said radioactivity detector;
(v) means for introducing said agent solution into said first conduit disposed in said first conduit;
(vi) a second conduit for introducing said agent solution into said introducing means;
(vi) pump means for pumping said agent solution; and
(vii) an optionally present UV-visible absorbance detector;
b) pumping said eluate from said chromatography column into said radiation detector so that a part of said radiation detection area of said flow cell is occupied by said eluate;
d) stopping said flow of said eluate;
e) pumping a volume of said agent solution through said second conduit and said means for introducing said agent solution into said first conduit, said volume of agent solution being sufficient to ensure that said eluate in said first conduit, said introducing means and said flow cell preceding said radiation detection area is placed into said radiation detection portion of said flow cell;
f) counting the radioactivity residing in the radiation detection portion of the flow cell in a static fashion;
g) flushing at least said radiation detection area of said flow cell with said agent solution; and
h) optionally repeating steps (b) through (h).
In some embodiments, said second conduit comprises means for the introduction of a gas into said second conduit. In other embodiments, the foregoing methods further comprising the step of flushing at least said radiation detection area of said flow cell with said gas, said gas flushing step being performed either immediately before or immediately after step (g).
In some embodiments, said means for the introduction of a gas into said second conduit is a tee. In other embodiments, said agent solution is a solvent.
In some embodiments, steps d-g are performed for every fraction eluted from said chromatography column. In other embodiments, steps d-g are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps d-g are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided by the present invention are improvements to a stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a sample solution containing at least one radiolabeled species and an agent solution into a means for mixing said sample solution and said agent solution to produce a mixed sample-agent solution;
(b) flowing said mixed sample-agent solution from said mixing means through a conduit and into a flow cell having a radiation detection area for detection of radioactivity;
(c) stopping said flow of said mixed sample-agent solution; and
(d) counting the radioactivity of said mixed sample within said radiation detection area;
the improvement comprising:
after step (c) flowing a volume of said agent solution through said mixing means and into said conduit between said mixing means and said flow cell in an amount at least equal to the volume of said conduit and said mixing means, to ensure that said volume of mixed agent-sample solution residing in said conduit and said mixing means is placed in said radiation detection area prior to counting in step (d). In some embodiments, said method further comprises the step of:
(e) flushing said flow cell with said agent solution to remove said sample solution from said flow cell.
In some embodiments, said agent solution comprises a scintillant.
Also provided by the present invention are improvements to a stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a sample solution containing at least one radiolabeled species through a conduit and into a solid flow cell having a radiation detection area for detection of radioactivity, said conduit having interposed therein means for introducing an agent solution into said conduit;
(b) stopping said flow of said sample solution; and
(c) counting the radioactivity of said sample solution within said radiation detection area; the improvement comprising:
after step (b) flowing a volume of an agent solution through said introducing means and into said conduit in an amount at least equal to the volume of said conduit and said introducing means, to ensure that said volume of sample solution residing in said conduit and said introducing means is placed in said radiation detection area prior to counting in step (c). In some embodiments, the method further comprises the step of:
(d) flushing said flow cell with said agent solution to remove said sample solution from said flow cell.
In some embodiments, said agent solution is a solvent.
Also provided by the present invention are improvements to a stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a sample solution containing at least one radiolabeled species and an agent solution into a means for mixing said sample solution and said agent solution to produce a mixed sample-agent solution;
(b) flowing said mixed sample-agent solution from said mixing means through a conduit and into a flow cell having a radiation detection area for detection of radioactivity;
(c) stopping said flow of said mixed sample-agent solution; and
(d) counting the radioactivity of said mixed sample within said radiation detection area; the improvement comprising:
after step (d) flowing a volume of gas through said mixing means and into said conduit and said flow cell in an amount sufficient to remove said sample from said radiation detection area of said flow cell.
In some embodiments, the methods further comprise the step of:
(e) flushing said flow cell with said agent solution.
Also provided by the present invention are improvements to a stop-flow method for measuring the radioactivity of a radiolabeled sample solution, the method comprising:
(a) introducing a sample solution containing at least one radiolabeled species through a conduit and into a solid flow cell having a radiation detection area for detection of radioactivity, said conduit having interposed therein means for introducing an agent solution into said conduit;
(b) stopping said flow of said sample solution; and
(c) counting the radioactivity of said sample solution within said radiation detection area; the improvement comprising:
after step (b) flowing a volume of a gas through said introducing means and into said conduit and said flow cell in an amount sufficient to remove said sample from said radiation detection area of said flow cell.
In some embodiments, the methods further comprise the step of:
(d) flushing said flow cell with said agent solution to remove said sample solution from said flow cell.
In some embodiments, said agent solution is a solvent.
Also provided by the present invention are methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
(a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column producing an eluate;
(ii) a radioactivity detector comprising a flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iii) means for mixing said eluate and an agent solution to form a mixture thereof;
(iv) conduit means for directing said mixture into said flow cell; and
(v) means for introducing a gas into said flow cell; and
(vi) an optionally present UV-visible absorbance detector;
(b) flowing said mixture of said eluate and said agent solution through said conduit and into said radiation detection area of said flow cell;
(c) stopping said flow of said mixture;
(d) flowing a volume of said agent solution through said mixing means and into said conduit means in an amount at least equal to the total volume of said conduit, said mixing means and said flow cell preceding said radiation detection area, to ensure that said volume of mixed agent-sample solution residing in said conduit and said mixing means is placed in said radiation detection area;
(e) counting the radioactivity of said mixed sample within said radiation detection area; and
(f) flushing at least said radiation detection of said area flow cell with said gas to remove sample mixture therefrom; and
(g) optionally repeating steps (b)-(f).
In some embodiments, the methods further comprises the step of:
(h) flushing said flow cell with said agent solution.
In some embodiments, said step (g) is performed immediately preceding or immediately after step (f). In some embodiments, said agent solution is a solution comprising a scintillant.
In some embodiments, steps b-f are performed for every fraction eluted from said chromatography column. In other embodiments, steps b-f are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps b-f are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided by the present invention are methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector having a flow cell, said flow cell having an input, an output, and a radiation detection area;
(iii) a conduit for conducting eluate from said chromatography column, said conduit connecting said chromatography column and said input of said flow cell, said conduit having interposed therein means for introducing an agent solution into said conduit and mixing said agent solution with said eluate to produce a mixed eluate-agent solution;
(iv) means for flushing at least said radiation detection area with a gas to remove said eluate from said radiation detection area; and
(v) an optionally present UV-visible absorbance detector;
b) simultaneously flowing said eluate from said chromatography column and said agent solution through said mixing means, thereby flowing said mixed eluate-agent solution into said radiation detection area of said flow cell;
c) stopping said flow of said eluate;
d) flowing a volume of agent solution through said mixing means and said conduit that is at least equal to the volume of said conduit, said mixing means and said radiation detector prior to said radiation detection area, to ensure that said volume of mixed agent-sample solution residing in said conduit, said mixing means and said radiation detector preceding said radiation detection area is placed in said radiation detection area;
e) counting the radioactivity of said mixed eluate-agent solution in said radiation detection area in a static fashion;
f) flushing at least said radiation detection area of said flow cell with said gas to remove said eluate therefrom;
g) optionally flushing at least said radiation detection area of said flow cell with said agent solution; and
h) optionally repeating steps (b) through (g).
In some embodiments, said agent solution comprises a scintillant.
In some embodiments, steps b-g are performed for every fraction eluted from said chromatography column. In other embodiments, steps b-g are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps b-g are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided by the present invention are methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
(a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column producing an eluate;
(ii) a radioactivity detector comprising a solid flow cell, said flow cell having a radiation detection area from which radioactivity is counted;
(iii) conduit means for directing said eluate into said flow cell, said conduit means having interposed therein means for introducing an agent solution into said conduit;
(v) means for introducing a gas into said flow cell; and
(vii) an optionally present UV-visible absorbance detector;
(b) flowing said mixture of said eluate through said conduit and into said radiation detection area of said flow cell;
(c) stopping said flow of said mixture;
(d) flowing a volume of said agent solution through said introducing means and into said conduit means in an amount at least equal to the volume of said conduit means, said introducing means, and said radiation detector preceding said radiation detection area to ensure that said volume of eluate residing in said conduit means, said introducing means and said radiation detector preceding said radiation detection area is placed in said radiation detection area;
(e) counting the radioactivity of said mixed sample within said radiation detection area; and
(f) flushing at least said radiation detection of said area flow cell with said gas to remove sample mixture therefrom; and
(g) optionally repeating steps (b)-(f).
In some embodiments the methods further comprises the step of:
(h) flushing said flow cell with said agent solution.
In further embodiments, said step (h) is performed immediately preceding or immediately after step (f). In further embodiments, said agent solution is a solution comprising a scintillant.
In some embodiments, steps b-f are performed for every fraction eluted from said chromatography column. In other embodiments, steps b-f are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps b-f are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided by the present invention are methods for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a liquid chromatography column;
(ii) a radioactivity detector having a flow cell, said flow cell having an input, an output, and a radiation detection area;
(iii) a conduit for conducting eluate from said chromatography column, said conduit connecting said chromatography column and said input of said flow cell, said conduit having interposed therein means for introducing an agent solution into said conduit;
(v) means for flushing at least said radiation detection area with a gas to remove said eluate from said radiation detection area; and
(vi) an optionally present UV-visible absorbance detector;
b) flowing said eluate from said chromatography column through said conduit and into said radiation detection area of said flow cell;
c) stopping said flow of said eluate;
d) flowing a volume of agent solution through said introducing means and said conduit that is at least equal to the total volume of said conduit, said mixing means and said radiation detector preceding said radiation detection area, to ensure that said volume of eluate residing in said conduit, said mixing means and said radiation detector preceding said radiation detection area is placed in said radiation detection area;
e) counting the radioactivity of said mixed eluate-agent solution in said radiation detection area in a static fashion;
f) flushing at least said radiation detection area of said flow cell with said gas to remove said eluate therefrom;
g) optionally flushing at least said radiation detection area of said flow cell with said agent solution; and
h) optionally repeating steps (b) through (g).
In some embodiments, steps b-g are performed for every fraction eluted from said chromatography column. In other embodiments, steps b-g are performed only when a threshold amount of radioactivity is detected by said radioactivity detector. In other embodiments, steps b-g are performed only when a threshold amount of UV or visible light is detected by said UV-visible absorbance detector.
Also provided in accordance with the present invention is a system for measuring radioactivity in a liquid eluate from a chromatography column, the system comprising:
a transparent detection tube;
an inlet conduit for supplying eluate to the detection tube;
an eluate conduit for supplying eluate to the inlet conduit;
an eluate valve disposed in the eluate conduit, the eluate valve opening to enable flow of the eluate from the eluate conduit through the inlet conduit and into the detection tube in response to a signal from a controller;
an agent conduit for supplying a liquid agent to the inlet conduit;
an agent valve disposed in the agent conduit, the agent valve opening to enable flow of the agent from the agent conduit through the inlet conduit and into the detection tube in response to a signal from the controller; and
at least one photomultiplier tube having an element disposed proximate the detection tube for measuring a parameter related to radioactivity of the eluate;
the controller being in informational communication with the eluate valve, the agent valve, and the photomultiplier tube to facilitate sequentially a flow stage, a push stage, a measuring stage, and a flush stage such that:
each one of the eluate valve and the agent valve are open to define the flow stage during which the eluate and the agent flow into the inlet conduit and into a portion of the detection tube;
the eluate valve is closed and the agent valve is open to define the push stage during which the eluate disposed in at least a portion of the inlet conduit is forced into the detection tube by the agent;
each one of the eluate valve and the agent valve is closed to define the measuring stage during which the eluate in the detection tube is substantially stationary, whereby the radioactivity measurement is performed on substantially stationary eluate for a time period; and
the eluate valve is closed and the agent valve is open to define a flush stage in which the detection tube is flushed of the eluate by a flow of the agent, whereby measurement of the radioactivity is improved by removing residual eluate from the detection tube,
whereby subsequent to the flush stage the system is in condition to initiate another flow stage.
In some embodiments, the foregoing system further comprises an eluate pump disposed in the eluate conduit for pumping the eluate and an agent pump disposed in the agent conduit for pumping the agent. In some embodiments, the detection tube is a coil.
In further embodiments, the eluate inlet and the agent inlet form a pair of inlet legs of a tee connector, and the inlet conduit form an outlet leg of the tee connector.
In still further embodiments, the agent includes a scintillant that produces flashes in response to receiving radiation from the eluate, whereby the photomultiplier tube senses the flashes to measure the radioactivity of the eluate.
In still further embodiments, the system further comprising a scintillant disposed outside of the detection tube, the scintillant produces flashes in response to receiving radiation from the eluate through the detection tube, whereby the photomultiplier tube senses the flashes to measure the radioactivity of the eluate.
In still further embodiments, the system further comprising a gas source in flow communication with the agent conduit and a gas valve for controlling the flow of gas into the agent conduit, the gas valve opening to substantially purge the detection tube of liquid, thereby augmenting the flush stage.
In some embodiments, the gas valve is open prior to the flush stage. In further embodiments, the gas valve is open subsequent to the flush stage. In further embodiments, the gas valve remains continuously opened during purging. In still further embodiments, the gas valve is cycled during purging to provide gas pulses.
In still further embodiments, the system further comprising a gas source in flow communication with the detection tube and a gas valve for controlling the flow of gas into the agent conduit, the gas valve opening to substantially purge the detection tube of liquid, thereby augmenting the flush stage, the gas source having an inlet in the detection tube to define a purgeable portion of the detection tube and an unpurgeable portion of the detection tube, the purgeable portion of the detection tube being substantially purged of liquid upon the opening of the gas valve.
In still further embodiments of the system, eluate disposed in the unpurgeable portion of the detection tube is pushed to a distal end of detection tube during the push stage so as to be disposed in the detection tube during a second measuring stage.
Also provided by the present invention is a system for measuring radioactivity in a liquid eluate from a chromatography column, the system comprising:
a transparent detection tube;
an inlet conduit for supplying eluate to the detection tube;
an eluate conduit for supplying eluate to the inlet conduit;
an eluate valve disposed in the eluate conduit, the eluate valve opening to enable flow of the eluate from the eluate conduit through the inlet conduit and into the detection tube in response to a signal from a controller;
an agent conduit for supplying a liquid agent to the inlet conduit;
an agent valve disposed in the agent conduit, the agent valve opening to enable flow of the agent from the agent conduit through the inlet conduit and into the detection tube in response to a signal from the controller;
at least one photomultiplier tube having an element disposed proximate the detection tube for measuring a parameter related to radioactivity of the eluate; and
a gas source for purging at least a portion of the detection tube of liquid; whereby the eluate is stopped in the detection tube for a time period during which radioactivity measurements are performed, the gas source purging at least a portion of the detection tube subsequent to such radioactivity measurements.
In some embodiments of the system, the gas source includes a gas inlet disposed in the agent conduit, whereby at least a portion of the inlet conduit and the detection tube are purged upon opening of the gas valve.
In further embodiments, the gas source includes an inlet disposed within the detection tube to define a purgeable portion of the detection tube and an unpurgeable portion of the detection tube, the purgeable portion of the detection tube being substantially purged of liquid upon the opening of the gas valve.
In still further embodiments, eluate disposed in the unpurgeable portion of the detection tube is pushed to a distal end of detection tube so as to be disposed in the detection tube during a second measuring stage.
In further embodiments, the present invention provides improvements to flow cells for the detection of a radioactive species in a liquid sample, wherein said flow cell has a tubular means for conducting said liquid sample through said flow cell; the improvement comprising a grounding wire disposed within said tubular means in the form of a helix or coil.
In still further embodiments, the invention provides a flow cell for detection of a radiolabeled species in a liquid sample, said flow cell comprising:
i) an exterior surface and an interior surface, said interior surface defining said radiation detection area;
ii) an inlet through-aperture;
iii) an exit through-aperture; each of said inlet and exit through-apertures connecting said radiation detection area with said exterior surface;
iv) tubular means entering through said inlet through-aperture, and exiting through said exit through-aperture, and directing flow of said eluate through said radiation detection area; and
v) a grounding wire interposed within said tubular means, said grounding wire being in the form of a helix.
In some embodiments of each of the foregoing methods, systems and flow cells of the invention, the flow cell can be either a liquid flow cell or a solid flow cell. Preferably, in some embodiments of each of the methods and systems of the invention the flushing gas is an inert gas, preferably nitrogen or helium.
In some embodiments of each of the foregoing methods of the invention, flushing with said gas comprises directing a steady stream of gas. In other embodiments of each of the foregoing methods of the invention, said flushing with said gas comprises directing a discontinuous stream of gas. In other embodiments of each of the foregoing methods of the invention, said flushing with said gas comprises flushing with pulses of gas. In one particularly preferred embodiment of each of the foregoing methods of the invention, the flushing with gas in the methods of the invention comprises the steps of:
i) flushing said flow cell with a first fluid;
j) optionally flushing said flow cell with a second fluid; and
k) optionally flushing said flow cell with a third fluid;
wherein each of said fluids is independently a gas, a solvent or an agent solution. In some embodiments, said first fluid and said third fluid are each an inert gas, and said second fluid is a solvent or an agent solution, and in other embodiments said first fluid and said third fluid are each a solvent or an agent solution, and said second fluid is an inert gas.
In some embodiments of the methods and systems of the invention, the liquid chromatograph will include a UV-visible absorbance detector.
In some embodiments of the methods and systems of the invention, the agent solution comprises a scintillant.